Simultaneous radio immunoassay of multiple antigens and _assay for cocaine metabolites

ABSTRACT

This invention includes an antigenic conjugate of ecgonine and a carrier which elicits anti-benzoylecgonine (cocaine metabolite) serum useful for the detection of cocaine metabolites in human organs and body fluids by immunoassay. The invention also includes the method of producing the anti-benzoylecgonine serum, the immunoassay method and composition including the anti-benzoylecgonine serum and a labeled benzoylecgonine and a non-labeled benzoylecgonine. 
     The invention also includes a composition comprising a plurality of antigenic conjugates which elicit a multivalent anti-serum capable of complexing a plurality of antigens in a multiple immunoassay detection method. The invention includes the method of producing the multivalent anti-serum and an immunoassay method employing the multivalent anti-serum. The invention further includes a composition comprising the multivalent anti-serum and a plurality of labeled antigens and non-labeled antigens.

This is a division of application Ser. No. 761,078, filed Jan. 21, 1977.

BACKGROUND OF THE INVENTION

The recent upsurge in the degree of drug abuse has led to the necessityfor rapid, reliable and efficient methods for detecting the presence ofdrugs, their metabolites or derivatives in human body fluids such asplasma, urine, etc. In recent years, numerous methods have beendeveloped for the detection of a variety of drugs subject to abuse, suchas morphine, barbiturates, amphetamines, lysergide, nicotine,diphenylhydantoin, chloradiazepoxide and cannabinoids.

The following patents describe these methods: U.S. Pat. No. 3,766,162,Spector; U.S. Pat. No. 3,775,536, Spector et al; U.S. Pat. No.3,799,741, Williams; U.S. Pat. No. 3,882,245, Spector et al; U.S. Pat.No. 3,843,696, Wagner et al; U.S. Pat. No. 3,853,987, Dreyer; U.S. Pat.No. 3,867,366, Rubenstein et al; U.S. Pat. No. 3,878,187, Schneider etal; U.S. Pat. No. 3,879,262, Schuurs et al; U.S. Pat. No. 3,884,898,Schneider; U.S. Pat. No. 3,888,864, Cleeland et al; U.S. Pat. No.3,952,091, Grunberg et al; U.S. Pat. No. 3,966,744, Goldstein et al;U.S. Pat. No. 3,888,866, Lente et al; and, U.S. Pat. No. 3,690,834,Goldstein et al.

One of the most convenient, efficient and accurate methods for thedetection of small amounts of organic compounds in fluids is theso-called radioimmunoassay method. See Radioimmunoassay Methods,Churchill, Livingston, London, 1971, and Yalow et al, Journal ofClinical Investigation, Vol. 39, pg. 1157, 1960.

Radioimmunoassay (RIA) is a term employed to describe any of severalmethods for determining small concentrations of substances in biologicalfluids based on the utilization of radioactively labeled substanceswhich form immuno-chemical complexes with antibodies to that substance.Various RIA techniques are known for measuring concentrations of bothantibodies and substances for which there exist antibodies. The RIA of asubstance for which there exists antibodies specific to that substanceis based on the observation that a known amount of that substance whichhas been radioactively labeled will compete equally with an unknownamount of that substance which is unlabeled for a limited number ofcomplexing sites on antibodies specific to the substance.

RIA is especially suited for the detection of extremely minute amountsof substances in biological fluids and would, therefore, be extremelyvaluable in the detection of drugs or their metabolites in thebiological fluids of the body. With conventional analytical methods, itis extremely difficult to measure the minute amounts of such metaboliteswhich occur in human body fluids due to the intake of the drugs.

As noted above, RIA procedures are based upon the characteristics thatan antibody binds equally to labeled or unlabeled antigen. Theconcentration of the non-labeled form in the solution determines therelative amount of labeled or non-labeled antigen which will bind toantibody. By maintaining the concentration of antibody and labeledantigen constant and conducting the RIA procedure using a series ofknown amounts of non-labeled antigen, a standard curve can beconstructed. Subsequently, when an unknown amount of antigen in a serumor other biological sample is reacted in the same way, its concentrationcan be determined by relating the value obtained to the standard curve.

The technique may be represented by the following scheme: ##STR1##where, Ag is the antigen to be assayed

Ag* is the labeled antigen and

Ab is the anti body

The amount of Ab is arranged to be insufficient to react with all ofAg+Ag*. The amount of Ag* added and Ag present in the standards or theunknown sample will compete for the limited binding sites on Ab. If Ag*is separated from Ag*Ab in the above reaction and the level of activityof each separated part measured, then their individual values or theirratios would be related to the amount of Ag present in the standards orthe sample. By the use of standard preparations of Ag, a calibrationcurve is generated.

To date, however, no completely satisfactory method has been proposedfor the radioimmunoassay of cocaine, its derivatives or metabolites inbody fluids.

Recent studies have shown that cocaine is rapidly metabolized in man.The principal metabolite that appears in urine is benzoylecgonine (BE)which is found along with smaller amounts of ecgonine (E). Cocaineitself is found in very low concentrations, if at all. Cocaine and itsmetabolites have been analyzed by thin-layer (TLC) and gaschromatography (GLC) and more recently by the Enzyme MultipliedImmunoassay Technique (EMIT). U.S. Pat. No. 3,888,866 describes a spinlabel immunoassay for cocaine derivatives.

The major disadvantages associated with TLC, GLC and EMIT assays aretheir lack of sensitivity and their lack of susceptibility toautomation. Moreover, EMIT assays are subject to interference due toenzyme poisons present in many biological specimens, particularly,endogenous lysozyme in some human urine specimens.

A further problem which confronts the art is the screening of bodyfluids for the presence of multiple drugs. If a subject is suspected ofhaving ingested a drug and no clue whatsoever exists as to the nature ofthe drug, it could be highly advantageous to be able to "screen" thesubject's body fluids for a plurality of drugs in a single test, therebyeliminating the necessity of having to run a series of tests designedonly to test for the presence of a single drug. Obviously, the provisionof a method for at least qualitatively analyzing a body fluid for thepresence of multiple drugs, thereby eliminating the presence of a largenumber of other drugs, would be more economical and of great benefit incontrolling and monitoring of drug abuse.

Immunoassay methods have been proposed for detecting the presence ofmultiple drugs in body fluids; however, these methods depend upon theproduction of an anti-serum for each particular drug in a separate donoranimal and combining the anti-sera in vitro to produce the necessaryreagent for the immunoassay method. To date, no method has been providedwhich enables the production of an anti-sera for a variety of drugsand/or their derivatives from a single donor animal thereby alleviatingthe disadvantageous time requirements required for the prior art method.Moreover, the maintenance of large numbers of animals and the attendantexpense would be eliminated if the anti-serum were derived from a singleanimal.

SUMMARY OF THE INVENTION

The present invention is predicated on the discovery that an antigenicconjugate of ecgonine or its acetate and a peptide, protein orpolysaccharide elicits anti-benzoylecgonine serum upon immunization ofan animal species therewith, said anti-serum being suitable for use in araioimmunoassay method for the detection of the cocaine metabolitebenzoylecgonine in organs and body fluids, particularly human urine.

The invention includes the antigenic conjugates of ecgonine or itsacetate and carrier (protein, peptide or polysaccharide); the anti-serumproduced by immunization of an animal with said conjugate and the methodof preparation of the anti-serum; an immunoassay for cocaine metabolitesemploying the anti-serum and compositions comprising the anti-serum andlabeled antigen (e.g., I¹²⁵ benzoylecgonine) and/or non-labeledbenzoylecgonine.

In another embodiment of the invention, anti-sera for a variety of drugsor drug metabolites are produced from a single animal which may beutilized in a method for the screening of multiple drugs (or theirmetabolites) in biological specimens. This embodiment avoids the"dilution effect" inherent in prior art methods where different specificanti-sera are produced in a number of different animals and thencombined for utilization in a screening method.

When the separately produced anti-sera are combined to prepare apolyvalent anti-serum, each is "diluted" by the other thereby lesseningthe sensitivity of the overall combined reagent.

According to this embodiment of the invention, an anti-serum againstseveral drugs (or their metabolites) is prepared from the same animaland utilized in, for example, a polyvalent radioimmunoassay PV-RIAcapable of screening biological specimens for a number of drugssimultaneously in a single assay. Inasmuch as the anti-sera are producedin situ in a single operation, the above-referred to "dilution effect"is avoided.

The invention includes a composition comprising the plural antigenicconjugates; the anti-serum produced by immunization of an animal withthe plural conjugates and the method of preparation of the anti-serum;an immunoassay for a plurality of antigens employing the anti-serum andcompositions comprising the anti-serum and the corresponding labeledantigens and/or non-labeled antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are inhibition curves;

FIGS. 3, 6 and 7 are calibration curves;

FIG. 4 shows cocaine metalolite levels;

FIG. 5 shows percent binding.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is predicated on the discovery thatimmunogens prepared by the coupling of ecgonine or its acetate toproteins, peptides or polysaccharides elicit anti-benzoylecgonine serumwhich is highly specific to benzoylecgonine in an assay for the cocainemetabolite in biological fluids, particularly human urine, therebyproviding a highly accurate, efficient and valuable tool for screeningsubjects for cocaine abuse.

It is most surprising that the ecgonine (or acetate)-carrier immunogenprovides an anti-benzoylecgonine serum which is more specific tobenzoylecgonine than ecgonine or any of the other cocaine metabolites.It is even further surprising that the ecgonine-carrier immunogenproduces a more highly reactive and specific anti-benzoylecgonine serumthan the benzoylecgonine-carrier immunogen. Although not wishing to bebound by any theory or mechanism, it is hypothesized that the greateractivity to benzoylecgonine suggests a possible conversion of asignificant portion of the conjugated ecgonine in the immunogen in vivoto a compound or compounds conformationally more closely related tobenzoylecgonine against which the major portion of the total antibodiessubsequently elicited was directed.

The antibody elicited by the ecgonine-carrier immunogen recognizesecgonine about one-half; benzoylnorecgonine about one-fourth; cocaine,nor-cocaine and ecgonine methylester less than about one-tenth asreadily as benzoylecgonine, the major cocaine metabolite. With continuedperiodic boosting, the degree of cocaine and ecgonine reactivityincreases but remains below that of benzoylecgonine.

A radioimmunoassay (RIA) method according to the present invention iscapable of detecting as little as 5-10 ng of benzoylecgonine, a majorcocaine metabolite in human urine, thereby rendering it the mostefficient and sensitive method for screening biological fluids forcocaine metabolites perfected to date. The technique enables themonitoring of cocaine intake by man in a relatively simple and highlyefficient manner.

It is preferred to employ sheep gamma globulin, bovine or porcinethyroglobulin as the protein carrier for the ecgonine in the preparationof the immunogen. However, it is to be understood that any suitableprotein, peptide or polysaccharide (amino sugar containingpolysaccharides) may be employed as the carrier.

Specific examples include: bovine, rabbit, pork, sheep or human serumalbumin; bovine, rabbit, pork, sheep or human gamma globulin;poly-L-lysine, hemocyanin, etc.

The ecgonine or acetate thereof may be bound to the protein, peptide orpolysaccharide carrier by reaction in a suitable reaction medium in thepresence of a suitable catalyst such as a water soluble carbodiimide;e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl,dicyclohexylcarbodiimide, etc.

The ecgonine or acetate forms an amide linkage through the 2-carboxylgroup on the ecgonine molecule with the amine groups of the proteins,peptides or amino sugar containing polysaccharides.

Where sheep gamma globulin is employed as the carrier and a carbodiimideas the catalyst, the reaction may be represented: ##STR2##

The anti-serum may be prepared by immunization of an animal with theantigenic conjugate followed by bleeding the animal and separating theanti-serum therefrom. Preferably, the antigenic conjugate is dissolvedin a saline solution to give a concentration ranging from 1.0 to 4.0mg/ml. Animals, e.g., horses, rabbits, sheep, goats, etc., may beimmunized by injection of the solution such that the animal receives adose of from about 2 to about 20 mg of the conjugate. Subsequentboosters of smaller amounts of the conjugate may be administeredaccording to any predetermined schedule. As noted above, it could not bepredicted that the anti-serum would be specific to benzoylecgonine asopposed to ecgonine from which the conjugate was prepared.

The anti-serum may be utilized in a radioimmunoassay method for thedetection of cocaine metabolites in organ homogenates and in biologicalfluids, particularly human urine. Standard calibration curves may beprepared employing known amounts of anti-serum, radiolabeledbenzoylecgonine and non-labeled benzoylecgonine. When utilizing theradioimmunoassay method for analyzing an unknown sample, the valuesobtained are compared with the calibration curves in order to ascertainthe amount of cocaine metabolite in the unknown sample.

It is to be understood that the invention includes compositionscomprising the anti-serum and varying amounts of the radiolabeledantigen or the non-labeled antigen for preparing the standardcalibration curves for utilization in the radioimmunoassay.

EXAMPLE 1 PREPARATION OF ANTI-BENZOYLECGONINE SERA Coupling of Ecgonineto Sheep Gamma-Globulin

Eighty mg of ecgonine (E) was added to a solution of 100 mg of sheepgamma-globulin (SGG) in distilled water and stirred until completelydissolved. The pH of the mixture was adjusted to 3.2 with dilute acidand 80 mg of water soluble 1-ethyl-3-(3-dimethylamino propyl)carbodiimide HCl was added. The final reaction mixture was incubatedovernight at room temperature (25° C.±2° C.) and then placed in adialysis bag and dialyzed at 4° C. for one week against two liters ofdistilled water changed 2-3 times daily. The contents of the bag wereassayed by EMIT and the remainder lyophilized.

Immunization, Boosters and Bleeding Schedules

The lyophilized immunogen, ecgonine-sheep gamma-globulin (E-SGG) wasdissolved in normal saline to give a solution of 4 mg/ml which was mixedwith an equal volume of Freunds' complete adjuvant and emulsified to athick paste using a blender. Two sheep were each injected with a totalof 5 ml of emulsion intramuscularly at four separate sites in the rumpand hip, and 2 ml were injected intramuscularly into each of twenty-fiveFlemish Giant Chinchilla rabbits. Subsequent primary and maintenanceboosters were given as described in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    IMMUNIZATION SCHEDULE OF SHEEP AND RABBITS FOR                                PREPARATION OF ANTI-BENZOYLECGONINE SERUM                                              Type Wt.         Total                                               Day                                                                              Recipient                                                                           Injection                                                                          Immunogen                                                                           Diluent                                                                             Volume Inj.                                                                          Route                                        __________________________________________________________________________    0  Sheep (S)                                                                           Primary                                                                            20 mg Complete                                                                            5.0 ml I.M.                                                             Freund                                                                        Adjuvant                                                     Rabbits (R)                                                                               2 mg Complete                                                                            2.0 ml I.M.                                                             Freund                                                                        Adjuvant                                                  21 S     Booster                                                                            10 mg Incomplete                                                                          5.0 mg I.M.                                                  I          Freund                                                                        Adjuvant                                                     R     Booster                                                                             1 mg       2.0 ml I.M.                                                  I                       and                                                                           S.C.                                         23 S     Booster                                                                            10 mg Incomplete                                                                          "      I.M.                                                  II         Freund       and                                             R           1 mg Adjuvant     S.C.                                         25 S     Booster                                                                       III                                                                     R           1 mg                                                           28 BLEED                                                                      __________________________________________________________________________

Maintenance boosters given at the previous dose at 4-6 week intervalsafter the third booster.

Anti-sera

The blood (100-200 ml for sheep and 20-30 ml for rabbits) was obtainedfrom the jugular vein of the sheep and the marginal car vein of rabbitsand held at 37° C. for 2-3 hours, then stored overnight at 4° C. Theserum was separated by centrifugation at approximately 3,000 rpm,decanted and stored at -10° C. until used. [It should be noted that thegamma globulin fraction of the anti-serum could be utilized to obtain arelatively more purified antibody].

EXAMPLE 2 Radioimmunoassay Antigens

One hundred microliters of I¹²⁵ -benzoylecgonine derivative (Sp.activity approx. 100 μCi/μg) (Hoffman-LaRoche, Inc., Nutley, N.J.) inphosphate buffered saline (PBS) at pH 7.2, containing 10,000 cpm/100 μlwas used in the binding assay. 0.2 ml of the radiotracer was used forqualitative assays and 0.5 ml for quantitative assays.

Radio-chromatography of Radiotracers

To ascertain the purity and chromatographic characteristics of theradioactive tracer, 1-10 μl of the concentrated solution was applied tosilica gel-G thin-layer plates. The plates were developed in an ethylacetate, methanol and ammonium hydroxide (170:20:10) solvent system--I(Davidow et al--American J. Clin., Pathol., 50, 714-719, 1968);chloroform, methanol and ammonia (75:25:5) solvent system--II andButanol; Acetic acid: Water (4:1:1) solvent III. To locate the variouscompounds the plates were sprayed first with silver nitrate (or acetate)solution followed by iodoplatinate spray reagent. After location ofcocaine (C), ecgonine (E), and benzoylecgonine (BE) on TLC plates, thetotal developed area was divided into seventeen equal horizontalsegments, scrapped individually from the TLC plate and counted forradioactive measurements using a Model MS-588 (Micromedic System, Inc.)gamma counter.

Radioimmunoassay

One-tenth ml aliquots of anti-sera diluted from 1:2 to 1:100 wereincubated with I¹²⁵ benzoylecgonine derivative representingapproximately 10,000 cpm/100 μl in the presence of either 0.1 ml ofnormal sheep serum (NSS) or normal rabbit serum (NRS) and sufficient PBS(ph 7.2) was added to each mixture to effect a final volume of 0.5 ml.The mixtures were then incubated at 25° C. for one hour and 0.5 ml of aneutral saturated ammonium sulfate solution was then added to all tubes.The tubes were vortex mixed and the mixture reincubated at 25° C. for 15minutes. The precipitates obtained after centrifugation at 3,000 rpm for30 minutes were washed twice with equal volumes of 50% saturatedammonium sulfate. The percent binding of the radioactive tracer was thendetermined. The dilution of sera from sheep and rabbits that gave 50-70%binding of the radiotracer was selected for the preparation of thestandard curve and the screening of biological specimens.

For qualitative screening of human urine specimens, 0.2 ml of theappropriate anti-serum dilution, 0.2 ml of the radiotracer, and 0.1 mlof the varying dilutions of benzoylecgonine, benzoylnorecgonine,ecgonine, ecgonine methylester, cocaine, nor-cocaine, or unknownbiological specimen (urine, plasma, organ homogenate) were used in theradioimmunoassay. For quantitative analysis of plasma, urine, or tissuespecimens from rabbits and rats, the volume of anti-serum dilution andradioactive tracer was increased to 0.5 ml and the volume of saturatedammonium sulfate to be added was increased to 1.0 ml. This latterprocedure provided a standard curve with an extended range of linearityover a concentration range of 0-1000 ng/ml, in contrast to the limitedlinearity (0-100 mg/ml) for qualitative screening.

Animal Experiments

Control plasma specimens from the marginal ear vein of four New ZealandAlbino rabbits which were approximately 3 kg in weight were collected.Two ml of a solution of cocaine hydrochloride (2 mg/ml) in saline wasthen injected I.V. into each animal. Blood specimens were collectedalternately from the marginal ear veins every one to two hours for atotal of seven hours and finally at 24 hours for the determination ofplasma levels of cocaine metabolites. Four mg of cocaine hydrochloridewas administered I.V. to another group of two anesthetized rabbits andhourly urine specimens collected directly from ureters for a total of 6hours.

Fifty and 20 mg of cocaine hydrochloride were injected I.V. and I.P.into two rabbits and two rats, respectively. After 4 hours the animalswere sacrificed and various tissues obtained for analysis.

Human Urines From Drug Abuse Programs

Urines from various clinics located in different parts of the City ofNew York were routinely analyzed by thin layer chromatograph (TLC). Allpositive urines for cocaine metabolite by TLC (total specimen 438) andapproximately an equal number of randomly obtained TLC negative urines(total specimens 615) were subsequently examined by the EMIT-Cocainemetabolite test and by radioimmunoassay.

Radiochromatography

Mobility (RF) of unlabeled cocaine, nor-cocaine, benzoylecgonine andecgonine in solvent system I was 0.91, 0.91, 0.05 and 0.00; in solventsystem II 0.93; 0.96, 0.48, and 0.08 and in solvent system III--0.38,0.51, 0.34 and 0.06, respectively. For I¹²⁵ benzoylecgonine derivativeover 80% of the radio-activity was at the RF 0.05, 0.08 l and 0.35-0.38in the three solvent systems, respectively. These results defined thechromatographic characteristics of I¹²⁵ -benzoylecgonine derivative(Roche Diagnostics).

Antibody Production

All rabbits (25) and both sheep produced anti-sera capable of bindingI¹²⁵ -benzoylecgonine derivative. The titers obtained in the firstbleeding of sheep were quite low while the titers in rabbits on firstbleeding were somewhat higher than the sheep. As the immunizing boosterswere continued the titers gradually rose until a 1:200 dilution forsheep and 1:500 for rabbit anti-sera were capable of binding 50% of I¹²⁵-benzoylecgonine derivative. Most of the studies reported are withanti-sera which had a titer of over 1:50 for 50% binding. It wasobserved that relatively steeper slopes of the standard curve areobtained if one works with anti-sera dilution giving 60-70% binding ofthe radioactive tracer.

The above procedure was repeated utilizing ecgonine acetate-sheep gammaglobulin conjugate and ecgonine acetate-bovine thyroglobulin conjugatein rabbits. Titers as high as 1:1000 were obtained.

Sensitivity of Radioimmunoassay

Typical standard curves obtained with varying concentrations of cocaine,benzoylecgonine, benzoylnorecgonine and ecgonine prepared in phosphatebuffered saline, pH 7.2, are shown in FIG. 1. From the figure it isevident that benzoylecgonine is the most efficient inhibitor for theassay. At least 2 and 5 times the concentration of cocaine and ecgonine,respectively, with benzoyl norecgonine occupying an intermediateposition, are required to produce the same amount of inhibition. Withperiodic boosting of the animals (e.g., rabbits), the relativeinhibition by cocaine and ecgonine increases but remains less than thatof benzoylecgonine. Additionally, the detection range ofbenzoylecgonine, the major cocaine metabolite, appears to be between2-10 ng/assay tube, the same as reported for most other drug RIAprocedures (FIG. 2).

FIG. 2 represents the binding inhibition of I¹²⁵ -benzoylecgoninederivative to ecgonine-SGG anti-serum by non-radioactive benzoylecgoninesample in 0.01 M phosphate-buffered saline, rabbit plasma and humanurine.

Normal rabbit serum and normal human urines are also seen to demonstrateapproximately 10 and 30% non-specific inhibition (FIG. 2). This isapparently due to endogenous materials present in these biologicalspecimens. The non-specific inhibition obtained with concentrated urinecan be reduced to less than 5% on dilution of the urine specimen 1:4with either water or normal saline.

FIG. 3 represents standard calibration curves for the detection ofbenzoylecgonine.

With benzoylecgonine a curvilinear relationship up to 100 ng/ml isevident with 0.2 ml each of the appropriate radiotracer and antibodydilution are used in conjunction with 0.1 ml of the standard solutions(FIG. 3). This type of standard curve was used for routine screeningpurposes. Beyond the 75 ng/ml concentration a gradual plateauing of thecurve is observed. However, the usable "quasi-linear" range cansubstantially be increased if the volume of the antibody and radiotracerare increased from 0.2 ml to 0.5 ml (FIG. 1). For quantitative analysistherefore it would be desirable to follow the latter procedure.

Specificity

As indicated above, the antibodies produced recognize benzoylecgonine(1.0) most readily followed by ecgonine (0.5) benzoylnorecgonine (0.25)and cocaine (0.15). Norcocaine and ecgonine methylester were leastreactive (less than 0.02). There was insignificant cross reactivity withatropine, homotropine and scopalamine which are somewhat structurallyrelated to the tropane portion of the cocaine molecule. A localanesthetic such as procaine, all other major drugs of abuse, includingamphetamine, barbiturates, morphine, methadone and about 30 othercommonly prescribed and used drugs were also examined and showedinsignificant cross reactivity.

Animal Experiments

FIG. 4 shows plasma cocaine metabolite levels in rabbits at various timeintervals after administration of 4 mg of cocaine-HCl intravenously. Thedata indicates that there is a rapid decline in plasma levels in thefirst two hours followed by a relatively slower decline. The plasmahalf-life of the metabolites in rabbits appears to be 1.8 l hr. and nonecan be detected in plasma after 24 hours when either 4 mg (FIG. 4) of 12mg of cocaine HCl are administered. The excretion of cocaine metabolitesinto the urine at various time intervals are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Excretion of Cocaine Metabolite(s) in Urine of Rabbits                        Receiving 4 mg. of Cocaine HCl Intravenously.                                         Vol.             μg    Total                                       Time (hr.)                                                                            of Urine μg/ml                                                                              Excreted/hr.                                                                           Excreted (μg)                            ______________________________________                                        Blank   31.0 ml  0.08*   2.48*    2.48*                                       1 hr.   7.40 ml  29.37   217.34   217.34                                      2 hrs.  5.93 ml  37.60   222.97   440.31                                      3 hrs.  5.73 ml  29.97   171.73   621.04                                      4 hrs.  6.93 ml  4.33    30.01    642.05                                      5 hrs.  18.50 ml 4.42    81.77    723.82                                      6 hrs.  9.45 ml  4.35    41.11    764.93                                      ______________________________________                                    

Biological background levels unrelated to cocaine or its metabolites.

From this data it is apparent that the rate of excretion peaks withinthree hours and then falls off gradually. The tissue distribution ofcocaine metabolites in rabbits and rats that received an overdose ofcocaine hydrochloride are presented in Table 3. Highest concentrationswere observed in blood, urine and kidney in the case of rabbits. Nourine was obtained from the rats. The highest concentration of cocainemetabolites were found in the blood, liver and kidney of rats withsmaller amounts in heart, lungs and brain. Higher concentrations ofcocaine metabolites were found in rat lung than in rabbit lungs andhigher concentrations of metabolites were also found in rat liver thanin the rabbit liver.

                  TABLE 3                                                         ______________________________________                                        Cocaine Metabolite(s) Distribution Levels in Organs                           and Biological Fluids of Rabbits and Rats 4 hrs. After                        Receiving 50 and 20 mg of Cocaine Hydrochloride I.V.                          and I.P. Respectively.                                                                 Radioimmunoassay                                                              Cocaine Metabolite(s) Levels μg/ml (gm)                           Organs     Rabbit        Rat                                                  ______________________________________                                        Urine      7.47          none obtained                                        Blood      8.35          4.00                                                 Liver      0.51          0.82                                                 Kidney     5.88          0.42                                                 Brain      0.10          0.09                                                 Heart      0.46          0.28                                                 Lungs      0.01          0.22                                                 ______________________________________                                    

Human Urines

The results from 1,053 selected urine specimens obtained from differentMethadone Maintenance Treatment Program and detoxification clinics inNew York City which were tested by various techniques for cocainemetabolites are summarized in Table 4. The total number of samples thatwere positive by all procedures was 399 (37.9%) whereas the total numberof specimens that were negative by all methods was 341 (32.4%). Therewere 84 (7.9%) samples which were positive with EMIT and RIA butnegative by TLC. Ninety-eight (9.3%) samples were positive by RIA butwere negative by EMIT and TLC. These differences are most probably dueto the relative lack of sensitivity of TLC and EMIT. In addition, therewere 27 (2.6%) samples which were positive by TLC and RIA but negativeby EMIT. These samples probably represent false negative findings forthe EMIT reagent. In addition 92 (8.7%) EMIT positive specimens werenegative by both TLC and RIA. Presumably these represent false positivefindings for the EMIT reagent. Only 8 (0.8%) of the specimens werenegative by RIA and positive by TLC and EMIT. These represent falsenegative findings for the RIA procedure. In addition 4 (0.4%) of thespecimens that were positive by TLC were negative by EMIT and RIA. Theseprobably represent false positive for the TLC procedure.

                  TABLE 4                                                         ______________________________________                                        Comparative Data on 1,053 Human Urines.sup.1 Analysed by TLC,                 Emit and Radioimmunoassay                                                     Total    Method of Analysis                                                   Samples  TLC      EMIT      RIA    % Total                                    ______________________________________                                        399      +        +         +      37.9                                       341      -        -         -      32.4                                       98       -        -         +      9.3                                        92       -        +         -      8.7                                        84       -        +         +      7.9                                        27       +        -         +      2.6                                        8        +        +         -      0.8                                        4        +        -         -      0.4                                        ______________________________________                                         .sup.1 Human urines were selected from Methadone Maintenance Treatment        Program clinics. All TLC positives and approximately equal number of TLC      negative specimens were picked up randomly.                              

The invention relates to a radioimmunoassay which is capable ofqualitatively and quantitatively determining cocaine metabolites inbiological specimens from humans as well as animals. Because the assaycan be performed directly on as little as 0.1 ml of specimen it issimple, rapid and adaptable to automation. It also readily lends itselfto the detection of cocaine abuse in man and is a sensitive analyticalmethod for animal experiments. Since the parent compound cocaine is notas readily recognized by the antibody as its metabolites, it is veryuseful for metabolic studies. From the foregoing data it is apparentthat the RIA procedure described here is a sensitive and specificscreening test which will serve as an efficient and reliable method fordetecting cocaine abuse.

There have been several prior attempts to prepare antibodies to cocainemetabolites and the degree of success has been relatively low whencompared with anti-sera prepared for other drugs. The present inventiondemonstrates that ecgonine-sheep gamma globulin conjugate is aneffective immunogen which elicits antibody production in most animalsimmunized. On the other hand, benzoylecgonine with SGG and other carrierproteins failed to produce antibodies in animals used in theseexperiments.

An unusual characteristic of the antibody produced is that it recognizesbenzoylecgonine more readily than it does ecgonine, which was used toproduce the anti-sera. It is conceivable that the animals converted asignificant portion of the injected hapten portion (ecgonine) of theconjugate (immunogen) to compounds which are conformationally moreclosely related to benzoylecgonine than to ecgonine. The total antibodypopulation thus produced is probably more closely attuned to the formerthan to the latter. Moreover, since there is a rapid and almost totalmetabolism of cocaine to BE and E in humans it is more useful to be ableto detect these metabolites than the parent compound.

ASSAY FOR DETECTION OF MULTIPLE ANTIGENS

In many cases of drug abuse, it would be desirable to conduct a singletest to screen for the presence of several drugs (or their metabolites)in a body fluid. Such a screening operation would operate to quicklynarrow the field so that quick and efficient quantitative tests can beconducted for a small number of drugs rather than conducting a largenumber of drug tests in a hit-or-miss operation.

According to a further embodiment of the invention, there is provided acomposition comprising a plurality of antigenic conjugates which form amultivalent anti-serum upon immunization of an animal therewith, thesaid multi-valent anti-serum being capable of complexing a plurality ofantigens in a simultaneous multiple immunoassay method.

In some instances, it is desirable to assay a biological fluid formultiple materials. For example, in screening subjects for suspecteddrug abuse, it would be highly desirable to conduct a singleradioimmunoassay method which would be capable of detectingsimultaneously the presence of two or more compounds which indicate thepresence of multiple drugs. Thus, a single test would be required ratherthan more expensive and time-consuming individual assays for each drugsuspected.

In prior art attempts to minimize costs and expedite the analysis ofspecimens antisera to single drugs have been combined in vitro toproduce a multivalent anti-serum. For example, an in vitro bivalentradioimmunosassay has been devised to screen for morphine andbarbiturates (Usategui-Gomez et al) Clin. Chem., 21, 1378-1382, 1975);Mule et al, Clin. Chem., 21, 81-86, 1975; and U.S. Pat. No. 3,952,091).These methods suffer from several disadvantages, however. Differentanimals are required for the production of each anti-serum, therebycontributing to the overall costs of the program. Moreover, the in vitromixing of the individual antisera must be carefully regulated to ensurethe production of multivalent antisera capable of giving reproducibleresults.

We have found that the immunization of a single animal with a pluralityof antigens enables the production of a multi-valent anti-serum which iscapable of complexing a plurality of antigens in a biological fluid.

For example, the antigenic conjugate composition may comprise two ormore of (1) an antigenic conjugate of ecgonine and a peptide, protein orpolysaccharide, (2) an antigenic conjugate of carboxylmethyl morphineand a peptide, protein or polysaccharide, (3) an antigenic conjugate of5-ethyl-5-(1-carboxyl-n-propyl) barbituric acid and a peptide, proteinor polysaccharide, (4) an antigenic conjugate of oxazepam and a peptide,protein or polysaccharide and (5) an antigenic conjugate ofnortriptyline or desmethyl imipramine and a peptide, protein orpolysaccharide. The multivalent anti-serum produced by the injection oftwo or more of these antigenic conjugates into a single animal would becapable of complexing two or more of the antigens: (1) benzoylecgonine,ecgonine and cocaine (cocaine and its metabolites), (2) morphine(opiates), (3) secobarbital (barbiturates), (4) diazepam or oxazepam(benzodiazepenes) and (5) amitryptyline (tricyclic anti-depressants)depending upon the particular antigenic conjugates injected into theanimal, in a multiple but simultaneous radioimmunoassay of the antigensin a biological fluid.

It has been found that the antigenic conjugates may be injectedsimultaneously or sequentially into the animal for eliciting theanti-serum. The anti-serum produced according to this method is usefulin a simultaneous multiple radioimmunoassay of a plurality of suspectedantigens in a biological fluid. The method is particularly valuable inthe detection of drug abuse and enables the screening of subjects for avariety of suspected drugs.

The anti-serum is combined with a plurality of radiolabled or non-labledantigens corresponding to the antigen to be detected to prepare standardcalibration curves for use in a radioimmunoassay. In the specificexamples set forth above, the radiolabeled antigens would comprise (1)I¹²⁵ -benzoylecgonine, (2) I¹²⁵ morphine, (3) I¹²⁵ secobarbital, (4)tritiated or I¹²⁵ diazepam, and (5) tritiated or I¹²⁵ amitriptyline ornortriptyline.

The invention also includes the anti-serum produced according to theabove-described method and compositions comprising the anti-serum and aplurality of the labeled antigens or a plurality of the non-labeledantigens for setting up calibration curves for employment in animmunoassay method. In the particular examples set forth above, thenon-labled antigens would comprise (1) a conjugate of ecgonine (oracetate) and a peptide, protein or polysaccharide, (2) a conjugate ofcarboxylmethyl morphine and a peptide protein or polysaccharide, (3) aconjugate of 5-ethyl-5-(1-carboxyl-n-propyl) barbituric acid and apeptide, protein or polysaccharide, (4) a conjugate of oxazepam and apeptide, protein or polysaccharide and (5) a conjugate of nortriptylineor desmethyl imipramine and a peptide, protein or polysaccharide.

In a typical experiment a group of rabbits were immunized with drugprotein conjugates of compounds related to morphine, barbiturate,cocaine, benzodiazepenes and tricyclic antidepressants. The "polyvalent"anti-sera elicited from the immunized animals were capable of bindingopiates, barbiturates, cocaine metabolites, benzodiazepenes andtricyclic antidepressants individually and/or in combination. Theanti-body titers for the various drugs ranged from 1:50 to 1:5000.

EXAMPLE 3 Preparation of Immunogens

One hundred mg of bovine serum albumin were dissolved in 20 ml ofdistilled water and 80 mg of carboxylmethyl morphine were added. Themixture was adjusted to pH 5.5 with dilute alkali and finally 80 mg ofwater soluble 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide was added.The mixture was incubated overnight at room temperature and the contentstransferred to an appropriate length of dialysis tubing and dialyzed at4° C. against 2 liters of distilled water with 2-3 changes each day. Thedialysate was removed from the bag after 5 days and used directly orlyophilized for future use. Prior to immunizations each conjugate wastested by RIA and EMIT for determining the degree of conjugation of thehapten with the protein. For the preparation of an immunogen tobarbiturates, rabbit serum albumin was substituted for the bovine serumalbumin and 5-ethyl, 5-(1, carboxyl-n-propyl) barbituric acid was usedas the hapten in the above procedure. Ecgonine was conjugated with sheepgamma globulin according to the method described in Example 1 to produceantibody to the cocaine metabolite, benzoylecgonine.

Antibodies to Benzodiazepenes were prepared: Ten mg of succinylatedoxazepam were dissolved in 5 ml of distilled water and 15 mg of bovinethyroglobulin were added until completely dissolved. The pH of themixture was adjusted to 4.9 and finally 20 mg of water soluble 1-ethyl-3(3-dimethylaminopropyl) carbodiimide HCl were added. The final reactionmixture was incubated overnight at room temperature and then placed in adialysis bag and dialyzed at 4° C. for 5 days against two liters ofdistilled water changed 2-3 times daily. The contents of the bag werelyophilized and 10 mg of the immunogen were dissolved in 1.25 ml ofnormal saline and mixed with an equal volume of Freund's completeadjuvant and emulsified to a thick paste using a syringe. Four rabbitswere each injected with a total 0.5 ml of the emulsion intradermallyinto multiple sites on the flank of the animal using approximately 50 μlat each site. Concomitantly with the immunogen, either 0.5 ml ofdiphtheria-pertussis-tetanus or pertussis vaccine was administeredseparately in a different site to enhance the immunologic response ofthe animals. Subsequent maintenance boosters were given every 6-8 weeks.Twenty to thirty ml of blood were obtained four weeks after the primaryimmunization from the marginal ear vein of rabbits and serum separatedfor titer determination. The succinylated derivative is employed toprovide carboxyl sites for conjugation. Any low molecular weightdicarboxylic acid may be employed, however.

Antibodies to tricyclic antidepressants were prepared: Fifty mg each ofnortriptyline hydrochloride (NT) and desmethylimipramine (DMI) weredissolved in 4 ml of dry pyridine and 20 mg of succinic anhydride wereadded with stirring until dissolved. The mixture was incubated overnightat room temperature followed by drying under a stream of nitrogen. Thetwo residues remaining were the succinylated derivatives and wereredissolved in 2 ml of dioxane each and 5-10 μl aliquots were examinedon fluorescent silica gel thin layer plates (TLC) for monitoring thedisappearance of the starting materials. In the case of succinylatednortriptyline the remaining portion was added to a separate solutioncomposed of 54 mg of bovine serum albumin (or bovine thyroglobulin)dissolved in 20 ml of a 0.05 M phosphate-buffered saline (PBS) pH 7.8.To this solution was added 190 mg of water soluble 1-ethyl-3(3-dimethylaminopropyl) carbodiimide HCL until dissolved. The finalreaction mixture was incubated overnight at room temperature. The freeand the conjugated succinylated derivative was separated by passing thesolution through a 25 cm, 1.5 cm diameter glass column filled with aslurry of sephadex G-50 (7 gm/100 ml PBS) and eluting with PBS. Two mlfractions were collected and protein peaks as measured at 260-280 nmwhich appeared in fractions 11-18 were collected for use to immunize theanimals. Eight fractions (total volume 16 ml) representing approximately50 mg of protein each from NT and DMI conjugates were lyophilized.

In case of succinylated DMI, approximately 50 mg were dissolved in 10 mlof distilled water and to this 50 mg of bovine thyroglobulin were added.The pH of the mixture was 5.7 and finally 60 mg of the above watersoluble carbodiimide was added. Incubation and dialysis was performedaccording to the method described above.

One and a half mg of the conjugate was dissolved in 0.5 ml normal salineand was mixed and emulsified with 0.5 ml of complete Freund's adjuvantand administered according to the technique as previously described. Theanimals were bled 4 weeks after the primary immunization via the centralear artery for determination of the individual titers. The firstbleeding was followed by intramuscular boosters of the respectiveconjugate dissolved in normal saline and administered every 4-6 weeksuntil desirable titers were obtained.

Again, any low molecular weight dicarboxylic acid may be substituted forthe succinic acid employed above.

Animals may be immunized with these drug-protein conjugates (immunogens)sequentially or simultaneously.

Immunization

One group of rabbits was sequentially immunized intramuscularly with 2mg of the individual immunogens and another group with the triple(morphine, barbiturate and cocaine) immunogens emulsified in completeFreund's Adjuvant. In the sequential method the second immunogen ws notadministered to the animal until the titer for the first one was atleast 1:10 (based on 50% binding for the respective radioactive tracer)and the third one was not injected until the "bivalent" anti-sera forfirst and second drug immunogens had a minimal titer of 1:10. A fourthimmunogen, namely, oxazepam, amitriptylene or desmethylimipramine wasadministered in a few selected animals having low titers for the firstthree drugs. In the simultaneous method, the triple immunogen wasadministered intradermally into multiple sites on the flank of theanimal approximately 50 μl at each site. Concomittantly with theimmunogen, 0.5 ml of DPT vaccine was also administered separately atdifferent sites to enhance the immunological response of the animals.Booster injections of 1 mg of the immunogens dissolved in phosphatebuffered saline were given at 6-8 weeks intervals after the primaryseries of injections. Diazepam immunogen was administered intradermallyat multiple sites into two of the rabbits to produce a tetravalentantibody. Pertussis vaccine (0.5 ml) was injected separately in othersites to enhance the immunologic response in these two animals. Forexample, FIG. 5 depicts two of the above-described immunizationschedules and drug combinations.

Iodinated (I¹²⁵)-labeled morphine, secobarbital, benzoylecgonine,tritiated diazepam amitriptyline or nortriptyline were used asradiotracers. When anti-sera that was capable of binding 2, 3 or 4different radioactively labeled drugs were obtained, individualcalibration curves were prepared for each drug.

FIGS. 6 and 7 show typical calibration curves for "tetravalent","trivalent", "bivalent" and "univalent" anti-sera. Because it ispossible to vary the relative ratios of the three iodinated antigens inthe triple assay, experiments were done to determine the appropriateratios of the antigens that had to be mixed to obtain similarcalibration curves.

Radioimmunoassay

For screening purposes equal volumes of the appropriate radiotracermixture and appropriate "polyvalent" antiserum dilution were mixed andstabilized overnight at 10° C. to give a "polyvalent" radioimmunoassay(PV-RIA) reagent. It is to be understood that overnight stabilization isnot essential; however, the slopes of the calibration curves obtainedwithout stabilization would vary from those obtained afterstabilization. Standard solutions ranging from 0-200 ng/ml of morphinesulfate, secobarbital, benzoylecgonine and diazepam were prepared inpooled normal urine. The standard solutions and 1,000 human urinespecimens obtained from the Methadone Maintenance Treatment ProgramClinics located in various boroughs of the City of New York wererandomly screened by the univalent, bivalent and trivalent assays usingan automated Micromedic High Speed Pippetting Station (ASP). Inaddition, 25 selected specimens were also screened by the tetravalentassay. Specimens showing disagreement of results between polyvalentassays were also tested with EMIT reagents. Four tenths ml of theunivalent or PV-RIA reagent and 0.1 ml of the undiluted urine specimenswere mixed by the APS and incubated for 30 minutes at room temperature.Five-tenths of a saturated ammonium sulfate solution was added to eachtube, vortex mixed and the mixtures reincubated for 15 minutes. Thesample tubes were then centrifuged for 15 minutes at 3,000 rpm and 0.5ml of the supernatant removed plus an equal volume of distilled water towash the residual radioactivity in the lines. The samples were thencounted on a Searle Analytical gamma counter (Model 1285-Z). The assaycould also be performed by mixing the biological specimen, antibody andthe radiotracer in sequence and incubating for 15 minutes. Incubationtime can be altered from a minimum of 15 minutes to a maximum of 60minutes.

The cut-off point (lowest drug concentration) for reporting a "positive"finding for the triple assay (FIG. 6-D) was at 4225 cpm (equivalent to75 ng/ml of barbiturate, 100 ng/ml of benzoylecgonine and 40 ng/ml formorphine). The same cut-off values were used for the univalent assay forcomparison purposes. FIGS. 6-A, B and C shows the calibration curves forvarious bivalent radioimmunoassays. The bivalent assays were performedprior to the induction of trivalent and tetravalent antibodies in thesame animals. For the quadruple assay (FIG. 7-B) 5500 cpm correspondingto approximately 25 ng/ml of morphine and barbiturate and 50 ng/ml ofbenzoyl ecgonine were used as a cut-off point for screening specimensfor these drugs. For diazepam, 10,540 cpm corresponding to about 50ng/ml were also used as a cut-off point for the detection of positivespecimens. It is evident from the calibration curves that ideally allthe calibration curves should have identical slopes and avidity forselecting a common concentration for cut-off values. However,calibration curves with different curvilinearity can also be used. Inthese cases, the detection limits for the various drugs will differ. Foran exclusion test, different cut-off points for various drugs would beconsidered acceptable. The results from univalent and trivalent assayswere compared to determine the performance of the latter (Table 5). Outof 1,000 specimens there were 14.5% false positives and 0.4% falsenegatives by the trivalent assay when compared with the results from theunivalent assay. For an exclusion test, these values are consideredwithin acceptable limits. The applicability of the PV-RIA procedure ascompared with univalent-RIA is demonstrated by a sensitivity of 99.6%[569/571×100] and a specificity of 85.5% [367/429×100]. As will be seenfrom results set forth below, a quadrivalent RIA gave equallysatisfactory results.

The same principle may also be applied to multiple immunoassays for avariety of antigens in other areas.

                  TABLE 5                                                         ______________________________________                                        Human Urines from 1,000 Subjects from Methadone                               Maintenance Treatment program and Detoxification Clinics                      Tested by Univalent and Trivalent Radioimmunoassays*                                  Univalent                                                                                 Cocaine           Trivalent                               No. of Samples                                                                          Barbiturate                                                                             Metabolite                                                                              Morphine                                                                              Assay                                   ______________________________________                                        367       -         -         -       -                                       12        +         +         +       +                                       9         +         +         -       +                                       36        +         -         +       +                                       109       -         +         +       +                                       66        +         -         -       +                                       70        -         +         -       +                                       267       -         -         +       +                                       Sub Total 569                                                                 62        -         -         -       +                                       1         +         -         -       -                                       1         -         +         -       -                                       Sub Total 64                                                                  TOTAL 1,000                                                                   ______________________________________                                         *A sample was considered positive if it contained >75 ng/ml of                barbiturate, >100 ng/ml of cocaine and >40 ng/ml of morphine.            

EXAMPLE 4 Clinical Testing Procedures

The above 1,000 randomly collected urine specimens from the MethadoneMaintenance Treatment Program Clinics and Detoxification Clinics of NewYork City were also screened by routine thin layer chromatography.According to the Davidow procedure (Amer. J. Clin. Pathol., 50, 714-719,1968). Specimens showing disagreement of results between univalent andpolyvalent assays were also tested with EMIT reagents (Rubenstein et al,Biochem. Biophys. Res. Comm. 47, 846-851, 1972) for the presence ofopiates barbiturates and cocaine metabolites. The results were thencompared with those obtained according to the above univalent, bivalentand trivalent RIA procedures.

Twenty-five urines were selected based on their thin layerchromatography findings for a quadruple RIA test. Twelve specimenscontained only one of the four drugs, 11 contained two or more drugs andtwo contained none of these drugs.

Animal Organ Homogenates

Twelve rats weighing approximately 300 gm were injectedintraperitoneally with varying amounts of d-amphetamine, barbiturate,cocaine, methadone and diazepam either alone or in differentcombinations. Two of the animals received none of the above drugs. Twohours after administration of the various drug(s), the animals weresacrificed and various organs, urine and blood collected. Organhomogenates, blood and urines were subjected to the univalent andtrivalent (morphine-barbiturate-cocaine metabolite) RIA procedure. Itwas not possible to collect sufficient blood and/or urine on all theanimals for use in both univalent and trivalent assays.

As noted above, three hundred sixty-seven of the thousand specimens thatwere negative by all three univalent RIA techniques were also negativeby trivalent RIA. Five hundred sixty-nine specimens were positive by oneor more of the univalent assays and were also positive by the tripleassay. Of the remaining 64 specimens, 62 were negative by the individualRIA's but were positive by the trivalent RIA.

Out of these 62 specimens, 42 were also negative by EMIT and TLCprocedures, ten were positive by TLC, five were positive by EMIT andfive positive by both the EMIT and TLC procedures. There were twospecimens that were positive for the univalent RIA (one for barbiturateand one for cocaine metabolite) that were negative by trivalent RIA.These specimens would represent false negative for trivalent RIA. If thetrivalent assay is compared to the combined results of the univalentRIA, EMIT and TLC then only 42 false positive and no false negativeswere found.

The comparative results obtained with 25 selected specimens tested byunivalent and tetravalent are presented in Table 6. The data indicatesthat a tetravalent RIA for screening urine specimens for four drugssimultaneously is feasible.

                  TABLE 6                                                         ______________________________________                                        Comparison of Results on 25 Selected Human Urines from                        Drug Abuse Program Tested by Univalent and                                    Tetravalent Radioimmunoassays                                                 Univalent                                                                     No. of Barbi-  Cocaine   Mor-          Tetravalent                            Samples                                                                              turate  Metabolites                                                                             phine Diazepam                                                                              Assay                                  ______________________________________                                        2      -       -         -     -       -                                      3      +       -         -     -       +                                      3      -       +         -     -       +                                      2      -       -         +     -       +                                      4      -       -         -     +       +                                      2      +       +         +     -       +                                      3      +       -         +     -       +                                      2      +       +         -     -       +                                      3      -       +         +     -       +                                      1      -       -         +     +       +                                      ______________________________________                                         off Values: Barbiturate, Morphine 25                                          Cocaine metabolite, Diazepam = 50 ng/ml                                  

Animal Organo Homogenates

The results obtained in univalent and trivalent radioimmunoassaysperformed on organ homogenates and biological fluids of rats that hadreceived either one, two, three drugs or none at all are presented inTable 7. The data indicates that whenever a drug, either alone or incombination, was detected by the univalent radioimmunoassay, it was alsodetected by the trivalent procedure (i.e., animals 1 through 7). When nodrugs or non-related drugs were administered to rats, neither theunivalent nor the trivalent radioimmunoassay procedures were positive(i.e., animals 8 through 12). In a few instances where lower levels ofdrug were administered to animals (numbers 4, 5 and 7), univalent assayswere negative when the cut off values of 25 ng/ml morphine and 50 ng/mlfor cocaine were used. Had a cut off value of 12.5 ng/ml and 25 ng/ml,respectively, been used, these univalent assays would have beenconsidered positive.

    __________________________________________________________________________                              Trivalent                                               Drug and              Morph-   Drug and               Morph-              Animal                                                                            Dose    Spec-                                                                             Univalent Barb-                                                                              Animal                                                                            Dose    Spec-                                                                              Univalent Barb-               #   Administered                                                                          imen                                                                              Barb                                                                             Coc.sup.c                                                                        Morph                                                                             Coc.sup.c                                                                          #   Administered                                                                          imen Barb                                                                             Coc.sup.c                                                                        Morph                                                                              Coc                __________________________________________________________________________                Brain                                                                             -  -  -   -        Cocaine-HCl                                                                           Brain                                                                              +  -  -   +                       Cocaine-HCl                                                                           Kidney                                                                            -  +  -   +        (0.3 mg/kg)                                                                           Kidney                                                                             +  -  -   +                   1   (0.6 mg/kg)                                                                           Liver                                                                             -  +  -   +    5   +       Liver                                                                              +  -  -   +                               Blood                                                                             QNS                                                                              QNS                                                                              QNS +        Phenobarb-Na                                                                          Blood                                                                              QNS                                                                              QNS                                                                              QNS +                                                      (1 mg/kg)                                                                     Morphine-SO.sub.4                              Phenobarb-Na                                                                          Brain                                                                             +  -  -   +        (0.5 mg/kg)                                                                           Brain                                                                              -  -  -   +                   2   (2 mg/kg)                                                                             Kidney                                                                            +  -  -   +    6   +       Kidney                                                                             -  +  +   +                               Liver                                                                             +  -  -   +        Cocaine-HCl                                                                           Liver                                                                              -  +  +   +                               Blood                                                                             QNS                                                                              QNS                                                                              QNS +        (0.3 mg/kg)                                                                           Blood                                                                              QNS                                                                              QNS                                                                              QNS +                                                              Urine                                                                              QNS                                                                              QNS                                                                              QNS +                                                      Morphine-SO.sub.4                                      Brain                                                                             -  -  -   -        (0.3 mg/kg)                                                                           Brain                                                                              +  -  -   +                       Morphine-SO.sub.4                                                                     Kidney                                                                            -  -  +   +        Phenobarb-Na                                                                          Kidney                                                                             +  -  +   +                   3   (1 mg/kg)                                                                             Liver                                                                             -  -  +   +    7   (0.7 mg/kg)                                                                           Liver                                                                              +  +  -   +                               Blood                                                                             -  -  +   +        Cocaine-HCl                                                                           Blood                                                                              QNS                                                                              QNS                                                                              QNS +                               Urine                                                                             QNS                                                                              QNS                                                                              QNS +        (0.2 mg/kg)                                                                           Urine                                                                              QNS                                                                              QNS                                                                              QNS +                                                      Amphetamine-                                   Morphine-SO.sub.4                                                                     Brain                                                                             +  -  -   +    8   HCl     Brain                                                                              -  -  -   -                       (0.5 mg/kg)                                                                           Kidney                                                                            +  -  +   +        (0.1 mg/kg)                                4   +       Liver                                                                             +  -  -   +                                                       Phenobarb-Na                                                                          Blood                                                                             QNS                                                                              QNS                                                                              QNS +        Methadone-SO.sub.4                             (1 mg/kg)                                                                             Urine                                                                             QNS                                                                              QNS                                                                              QNS +    9   (0.6 mg/kg)                                                                           Kidney                                                                             -  -  -   -                                                  10  Diazepam                                                                      (0.04 mg/kg)                                                                          Liver                                                                              -  -  -   -                                                      Saline                                                                    11, 12                                                                            (no drugs)                                                                            Blood                                                                              QNS                                                                              QNS                                                                              QNS -                   __________________________________________________________________________     .sup.a Cut off Value: Barbiturate and Cocaine  50 ng/ml Morphine  25          .sup.b Sacrificed 2 hrs. after drug                                           .sup.c Cocaine metabolite measured as benzoylecgonine equivalents             QNS = quantity Not Sufficient to Test                                    

As noted above, the applicability of the polyvalent RIA procedure ascompared with the univalent procedure is demonstrated by a sensitivityof 99.6% (569/571×100) and a specificity of 85.5% (367/429×100). Thefalse positive and false negative rates, namely 14.5% and 0.4%,respectively, are considered within acceptable limits for screeningpurposes. It should be noted that most of the presumably false positivefindings may in fact be due to drugs which were below the cut off valuefor an individual drug but were above the trivalent RIA cut off value byan additive effect.

In most laboratories and major drug abuse programs, approximately 80% ofthe clinical specimens are negative for the major drugs subject toabuse. The polyvalent RIA described could serve as an exclusion testassay for such drugs. By eliminating approximately 80% of the specimensfrom further consideration, substantial savings in time and expensecould be achieved. The exclusion procedure could also be used inscreening forensic toxicology specimens where major drug classes couldbe ruled out by a simple polyvalent immunoassay screen of organhomogenates and biological fluids.

Even though it is possible to prepare a polyvalent antibody by in vitromixing of univalent antisera from different animals, the preparation ofa polyvalent antisera in the same animal produces more uniform antiseraand requires fewer animals for antibody preparation. Since fewer animalsare required, there is also less chance that the serum of a particularanimal in the pooled sera will cause untoward reactions. An additionaladvantage of the in vivo polyvalent anti-sera is that the potency of alow titered component(s), i.e., cocaine and/or diazepam, will not befurther reduced by in vitro mixing.

In sequential immunization procedures described above and depicted inFIG. 5 nearly all animals produced antibodies to both morphine andbarbiturates. Approximately one-half of the animals that had respondedto the two immunogens also responded to the third (i.e., cocainemetabolite) (FIG. 5A). Two of the rabbits that had responded to allthree immunogens were subsequently inoculated with the fourth (i.e.,diazepam) (FIG. 5B). At 1-2 or 1-5 dilution slopes of the calibrationcurves were adequate for use in quadruple RIA. Another group of twoanimals with morphine barbiturate-cocaine metabolite antibodies weresubsequently inoculated with the fourth (i.e., amitriptyline) immunogen.

Ideally each constituent of a polyvalent antiserum should have similarbinding properties so that a single dilution would be appropriate forall of the individual components (see arrows FIG. 5). For thesimultaneous "triple immunizations" three of four animals producedanti-sera capable of binding the three drugs to varying degrees,suggesting that both sequential and simultaneous immunization procedurescould be utilized.

Ideally the calibration curves for the various drugs should haveidentical slopes. FIGS. 6 and 7 show typical calibration curves forvarious polyvalent radioimmunoassays obtained by a single dilution ofthe various anti-sera. In general, the slopes of the individualcalibration curves in the multiple RIA's were flatter than if they wereprepared by single RIA's. In some animals it was not practical tooptimally dilute the antiserum for each drug, because the titers andslopes of the calibration curves for the three or four drugs weresignificantly different. The calibration curves obtained with thesereagents had somewhat different slopes. Consequently, the detectionlimits for the drugs varies. By appropriate scheduling of the boosterinjections, it is possible to obtain polyvalent antisera which wouldprovide optimal binding for all drugs at a single dilution.

Theoretically, the capability of animals to produce antibodies tomultiple antigens is unlimited as long as the antigens are notchemically related. For most purposes, however, it would not bepractical to utilize the same animal for more than about ten, preferablyfive, antigens.

The radiolabelled compounds utilized according to the present inventionmay be prepared according to well known and/or readily availablemethods. See, for example, Davis et al, Clin. Chem. 21/10, pp. 1498-1505(1975). See also ¹⁴ C, ³ H, ¹²⁵ I and ³⁵ S Labelled Drugs, New EnglandNuclear, September, 1976. It is also to be understood that drugs (orderivatives thereof) labelled with enzymes, free radicals, sensitizedred blood cells, etc. may also be employed in applicable immunoassays.

While the above description has been limited to a discussion ofradioimmunoassay procedures, it is to be understood that the principlesof this invention are applicable to other immunoassays such as EMIT,FRAT, HI, etc.

Reference may be had to the following for descriptions of variousmethods and reagents employed in practicing the present invention:Spector et al, Science, Vol. 168, pp. 1347-48, June 12, 1970; Spector etal, Science, Vol. 174, pp. 1037-1039, Dec. 3, 1971; Peskar, Jour. Pharm.Exp. Therap., Vol. 186, pp. 167-168 (1973); Dixon et al, Jour. Pharm.Sci., Vol. 64, pp. 937-38 (1975); Cleeland et al, Clin. Chem., 2216, pp.712-725, (1976); Aherne, Br. J. Clin, Pharmac. Vol. 3, pp. 561-65(1976); Satoh et al, J. Biochem., Vol. 73, pp. 1115-18 (1973); Satoh etal, J. Biochem., Vol. 75, p. 1302.

What is claimed is:
 1. In an immunoassay method for the detection of acocaine metabolite in a biological tissue or fluid wherein an anti-serumis combined with said biological tissue or fluid to form a complex withsaid cocaine metabolite, said complex being detectable in an assay, theimprovement comprising employing an anti-benzoylecgonine serum producedby immunizing an animal by injecting into said animal an antigenicconjugate of ecgonine or the acetate thereof and a peptide, protein orpolysaccharide and eliciting serum from said animal.
 2. The immunoassaymethod of claim 1 wherein said conjugate is a conjugate of ecgonine andsheep gamma-globulin.
 3. A composition suitable for use in animmunoassay method for detecting a cocaine metabolite in a biologicaltissue or fluid comprising an antibenzoylecgonine serum and a labeledbenzoylecgonine, wherein said anti-benzoylecgonine serum is produced byimmunizing an animal by injecting into said animal an antigenicconjugate of ecgonine or the acetate thereof and a peptide, protein orpolysaccharide and eliciting serum from said animal.
 4. The compositionof claim 3 wherein said labeled benzoylecgonine is I¹²⁵ -benzoylecgoninederivative.
 5. The composition of claim 4 wherein said anti-serum isproduced employing an antigenic conjugate of ecgonine or the acetatethereof and a gamma globulin or a thyroglobulin.
 6. The composition ofclaim 5 wherein said anti-serum is produced employing an antigenicconjugate of ecgonine and sheep gamma globulin.
 7. A compositioncomprising anti-benzoylecgonine serum and benzoylecgonine wherein saidanti-benzoylecgonine serum is produced by immunizing an animal byinjecting into said animal an antigenic conjugate of ecgonine or theacetate thereof and a peptide protein or polysaccharide and elicitingserum from said animal.
 8. The composition of claim 7 wherein saidanti-serum is produced employing an antigenic conjugate of ecgonine oran acetate thereof and a gamma globulin or a thyroglobulin.
 9. Thecomposition of claim 7 wherein said anti-serum is produced employing anantigenic conjugate of ecgonine and sheep gamma globulin.